Solarbio Albumin Content Assay Kit (Bromocresol Green Colorimetry)

Ikhtisar Produk

Product Components

Persiapan Solusi:

1. Color Development Solution:
   • Prepare the color development solution according to the number of samples by mixing Reagent One, Reagen Dua, and Reagent Three in the ratio of 10μL: 990μL: 10μL (1010μL for 5 tests). Mix thoroughly and use immediately.
2. Solusi standar:
   • Mempersiapkan a 10 mg/mL albumin standard solution. Sebelum digunakan, take 200μL of the 10 mg/mL albumin standard solution and add 200μL of the extraction solution to prepare a 5 mg/mL albumin standard solution. Mix thoroughly and use immediately.

Deskripsi Produk:

• Albumin:
  • The liver synthesizes the main protein in human plasma.
  • An important nutrient that maintains plasma osmotic pressure.
  • Binds with various nutrients, hormon, and drugs.
  • Reflects the nutritional status of the body.
  • Used to screen for diseases affecting liver metabolic function (misalnya, cirrhosis, liver damage, malnutrition, malignant tumors).
• Metode deteksi:
  • In a pH 4.2 buffer solution, serum albumin carries a positive charge.
  • In the presence of a non-ionic surfactant, albumin binds with the negatively charged dye Bromocresol Green.
  • Forms a blue-green complex.
  • The complex has an absorption peak at a wavelength of 630 nm.
  • Color intensity is directly proportional to the concentration of albumin.
• Reaction:
  • Albumin + Bromocresol Green → Blue-Green Complex (630 nm)

Catatan:

• Before the experiment, selecting 2-3 samples with expected large differences is recommended to conduct a preliminary experiment.
• If the sample absorbance is not within the measurement range, diluting or increasing the sample volume is recommended for detection.

Required Instruments and Supplies (to be prepared):

• Visible spectrophotometer/microplate reader
• Low-temperature centrifuge
• Keseimbangan analitik
• Microglass cuvettes/96-well plates
• Adjustable pipettes
• Mortar/homogenizer/ultrasonic cell disruptor
• Ice and distilled water

Prosedur:

Pemrosesan Sampel (The sample volume can be adjusted accordingly; specific ratios can be referenced from literature):

1. Sampel jaringan:
   • Add extraction solution in a ratio of sample mass (G) terhadap volume larutan ekstraksi (ml) dari 1:5~10 (recommend weighing 0.1g of the sample and adding 1.0mL of extraction solution).
   • Homogenize the sample in an ice bath, then centrifuge at 4℃, 8000g untuk 10 menit.
   • Discard the pellet and keep the supernatant on ice for testing.
2. Bacterial/Cell Samples:
   • Add extraction solution in a ratio of bacterial/cell count (10^6) terhadap volume larutan ekstraksi (ml) of 5~10:1 (recommend adding 1.0mL of extraction solution to 5 million bacteria/cells).
   • Disrupt the bacteria/cells in an ice bath using an ultrasonic cell disruptor (power 200W, ultrasonicate for 3s, interval 7 detik, waktu keseluruhan 5 menit).
   • Centrifuge at 4℃, 8000g untuk 10 menit.
   • Discard the pellet and keep the supernatant on ice for testing.
3. Liquid Samples:
   • Measure directly.
   • If the liquid is turbid, centrifuge and use the supernatant for measurement.

1. Preparation of Visible Spectrophotometer/Microplate Reader:
   • Preheat the visible spectrophotometer/microplate reader for at least 30 menit.
   • Set the wavelength to 630 nm.
   • Zero the visible spectrophotometer with distilled water.
2. Operation Table (Add the following reagents to micro glass cuvettes/96-well plates):

• Mix well and let stand at room temperature for 20 detik.
• Measure the absorbance of each tube at 630 nm, recording the values as A_test, A_standard, and A_blank.
• Calculate ΔA_test = A_test – A_blank, and ΔA_standard = A_standard – A_blank.
• Note that the blank tube and standard tube only need to be measured 1-2 waktu.

Catatan: The length of the standing time can affect the detection results. It is recommended to react directly in the micro glass cuvettes/96-well plates for 20 seconds and then measure the absorbance.

Albumin Content Calculation

1. Calculation by Sample Protein Concentration

Albumin content (mg/mg prot) = ΔA measured × (C standard ÷ ΔA standard) × V sample ÷ (V sample × Cpr) = 5 × ΔA measured ÷ ΔA standard ÷ Cpr

2. Calculation by Sample Mass

Albumin content (mg/g massa) = ΔA measured × (C standard ÷ ΔA standard) × V sample ÷ (W × V sample ÷ V sample total) = 5 × ΔA measured ÷ ΔA standard ÷ W

3. Calculation by Bacterial/Cell Number

Albumin content (mg/10^6 cells) = ΔA measured × (C standard ÷ ΔA standard) × V sample ÷ (V sample × N ÷ V sample total) = 5 × ΔA measured ÷ ΔA standard ÷ N

4. Calculation by Liquid Volume

Albumin content (mg/mL) = ΔA measured × (C standard ÷ ΔA standard) × V sample ÷ V sample = 5 × ΔA measured ÷ ΔA standard

Definitions:

• C standard: Standard tube concentration, 5 mg/mL;
• V sample: Sample volume added, 0.02ml;
• V sample total: Total extract volume added, 1ml;
• Cpr: Konsentrasi protein sampel, mg/mL;
• W: Sample mass, G;
• N: Total number of bacteria/cells, in 10^6

Catatan:

1. If ΔA_test is less than 0.010 or the absorbance of the test tube is close to that of the blank tube, you can increase the sample volume before measurement. If ΔA_test is greater than 0.5, it is recommended to appropriately dilute the sample supernatant with extraction solution before measurement. Note to simultaneously adjust the calculation formula.
2. If the sample becomes turbid after adding the color developer, it is recommended to appropriately dilute the sample supernatant with extraction solution before measurement. Note to simultaneously adjust the calculation formula.

Contoh Eksperimental:

1. Take 20μL of human serum sample, dilute it 10 times with extraction solution, follow the measurement steps, and measure it using a 96-well plate. Perhitungan: ΔA_test = A_test – A_blank = 0.426 – 0.124 = 0.302, ΔA_standard = A_standard – A_blank = 0.406 – 0.124 = 0.282. Calculated based on liquid volume: Albumin Content (mg/mL) = 5 × ΔA_test ÷ ΔA_standard × 10 (dilution factor) = 53.546 mg/mL.